We have developed a model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic (hyporesponsive) T cells. We call this state adaptive tolerance. We inject CD4+, cytochrome c-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse (called mPCC) expressing the cytochrome c antigen under the control of the MHC class I promoter and an immunoglobin heavy chain enhancer. Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. In vivo BrdU labeling shows a slow T cell turnover of about 5% per day. This hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. Interestingly, if the retransfer is into a host expressing the antigen, the cells remain hyporesponsive and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggests that the tolerance process is adaptable to different levels. During the past year we have completed a detailed biochemical analysis of the impairment in T cell receptor (TCR) signaling in the adaptively tolerant T cells. Compared to naive T cells, the tolerant T cells had normal levels of TCR and a 40% increase in CD4 expression. Following anti-CD4 crosslinking, LCK activity was slightly reduced in an in vitro kinase assay; however, following anti-TCR beta and anti-CD4 stimulation, TCR zeta chain phosphorylation was actually enhanced at high doses, possibly because the level of Fyn was also elevated in the tolerant cells. The predominant signaling block was at the level of ZAP-70 kinase activity, which was decreased 75% in vitro. Despite a compensatory increase in the amount of ZAP-70 protein, activation-induced phosphorylation of LAT and PLC-gamma1 were greatly reduced. The major impact of this impairment was on the calcium and NFkB pathways, with only a modest decrease in ERK1/2 phosphorylation. This state was contrasted with T cell clonal anergy in which the Ras/MAP kinase pathway was preferentially impaired and there was little or no block in ZAP-70 kinase activity, LAT and PLC-gamma1 phosphorylation, and NFAT activation. Both hyporesponsive states manifested a block in IkB degradation. These results demonstrate that T cell adaptive tolerance and clonal anergy are distinct biochemical states, possibly providing T cells with two molecular mechanisms to curtail responsiveness in different biological circumstances.